Luminescence detection and in particular fluorescence detection systems have been known for a long time in the prior art. In current analytical methods the photometric evaluation of analytical test elements is one of the most common methods for rapidly detecting or rapidly determining the concentration of analytes in samples. In general photometric evaluations are used in the field of analytics, environmental analytics and above all in the field of medical diagnostics. Test elements which are evaluated photometrically are very important especially in the field of blood glucose diagnosis from capillary blood.
In fluorescence-spectrometric detection systems, the intensity of the emission of the fluorescent substances is directly proportional to the intensity of the excitation. The intensity of excitation can be influenced by many factors for example by the behaviour of the light source over time or changes in the light paths. Changes in the light path can give rise to different excitation intensities especially in a small instrument which is not fixed in a laboratory for measurement. Hence it is very difficult to reproducibly carry out absolute fluorescence measurements.
For this reason current fluorescence test systems use a reference substance to which the test can be referred. Various types of reference substances are known.
For example it is known in the prior art that a second fluorophore which emits at a different wavelength to that of the specific fluorophore for the analyte can be used in detection systems which are based on a fluorophore. Hence this second fluorophore can be used as a reference. The two fluorophores can be distinguished by using two different filters (and two detectors) wherein one fluorophore is not chemically converted and can be used as a reference. This detection system is for example described in the following literature reference: Principles of Fluorescence Spectroscopy, J. R. Lackowicz, Kluver Academic/Plenum Publishers, New York, Boston, Dordrecht, Moscow 1999, 2nd edition.
Another approach is a time-resolved and phase-modulated referencing in which a very long-lived fluorophore which has a longer lifetime than a short-lived fluorophore that is specific for the analyte, is used as a reference substance. Whereas the parameters of the long-lived luminescence are not influenced by the analyte, the intensity of the short-lived luminophore which is specific for the analyte changes depending on the respective analyte concentration. Then time-resolved measurement allows firstly the analyte fluorophore to be measured and subsequently the decay of the reference is recorded. The ratio of the decay signal to the signal of the analyte plus that of the reference substance must always be the same independent of the intensity of the irradiation.
WO 99/06821, also published as U.S. Pat. No. 6,602,716 (B1), discloses such a system. In this case at least two different luminescent materials are used which are co-immobilized on a support, the first of which responds to the parameter of the analyte to be determined at least as regards luminescence intensity and the second of which does not respond to the parameter to be measured at least as regards luminescence intensity and decay time. The luminescent materials have different decay times. The time or phase behaviour of the resulting luminescence response can thus be used to generate a reference variable for determining the parameter of the analyte to be measured.
WO 02/056023 discloses an optical sensor for determining at least one parameter in a sample in which also in this case an indicator material which responds to the parameter with a short decay time and a reference material with a long decay time which does not respond to the parameter are used. The indicator material and the reference material are immobilized on a common support and are covered on the sample side with a light-impermeable layer.
However, referencing by means of a second fluorophore which is measured at a different wavelength requires a much more elaborate apparatus. Thus for example two filters are required instead of only a single filter in order to block the excitation light and usually two detectors are also necessary. Furthermore, due to the fact that the optical paths are separated after the excitation into different detectors, one of the two paths may be defective which would then result in an incorrect referencing.
A disadvantage of time-resolved or phase-resolved referencing is that the measurements of the analyte always have to be carried out against a luminescent background. This means that there is always a certain offset or signal background which limits the measuring range. This principle is shown for example in FIG. 1.
FIG. 1 shows the measuring range of the analyte signal with and without the reference. Without the reference fluorophore it is possible to optimally adapt the measuring range to the dynamic range that has to be spanned. With the reference fluorophore a part of the measuring range is used by the reference.
The reference substances or reference luminescent substances which are used in the above-mentioned methods of the prior art can only be used as a reference substance for comparison with the analyte but they cannot fulfil further functions.
However, an important issue when determining analytes in the diagnostic field is also detection of wetting and/or a filling control of the test field.
There are additional approaches in this field, such as, WO 83/00931, also published as U.S. Pat. No. 4,420,566, which discloses a device and a method for detecting a liquid sample in which it is determined whether an adequate amount of sample liquid has been applied. This is achieved by a drop detector which comprises a light source that projects radiation onto the sample, said radiation being within the absorption band of water. The sample carrier is firstly irradiated in a dry state and then in the wetted state and thus the moisture is measured. The moisture content of the sample is then calculated from the difference between the detected signals.
U.S. Pat. No. 5,114,350 discloses a method and a device for determining the concentration of an analyte in a sample of body fluid. The degree of wetting of the reaction support is measured by measuring the amount of reflected light which decreases with increasing wetting.
DE 10248555 A1, also published as US 2004/136,871, discloses a method for detecting and compensating for an under-dosing of test strips. A test element is described in this document which comprises an analyte-specific reagent which interacts with an analyte in a sample, and a control substance which interacts with a sample matrix of the sample. The analyte-specific reagent interacts with the analyte depending on the analyte concentration in a detection wavelength range. When the test field is irradiated, the control sequence interacts with the sample matrix as a function of the amount of sample applied to the test field. The control substance can also react with the water contained in the sample matrix. One example which is mentioned in DE 10248555 A1, also published as US 2004/136,871, uses chlorophenol red as an analyte-independent colour former for determining the amount of sample and 2,18-phosphomolybdic acid as an analyte-specific reagent for determining the glucose concentration in a sample e.g. a blood sample. Thus in the said example of this document two wavelength ranges have to be detected which complicates the apparatus for this test system.